This invention relates to the use of enzymes for protection of plants against phytopathogenic fungi. In particular, the invention relates to the use of enzymes showing esterase activity for protecting plants against phytopathogenic fungi by inhibition of fungal appressorium formation. In addition, the invention relates to the use of recombinant pepper esterase expressed by using Escherichia coli to protect plants against phytopathogenic fungi by inhibition of fungal appressorium formation.
Carboxylesterases are enzymes that catalyze the hydrolysis of compounds containing an ester bond. Genes encoding esterase or lipase enzymes have mainly been studied in mammals and microbes (Contreras et al. 1996; Feller et al. 1991; Kok et al. 1993; Langin et al. 1993; Osterund et al. 1997). Lipases in plants have been largely studied in germinating oil seeds where they provide energy for embryonic growth (Huang, 1987).
In plant-microbe interactions, a tobacco esterase gene, hsr203J, has been isolated from tobacco in a hypersensitive reaction against the pathogenic bacterium Ralstonia solanacearum (Badounin et al. 1997; Pontier et al. 1994). Recently, the EDS1 gene that is an essential component of the R gene-mediated disease resistance in Arabidopsis has been isolated and found to share homology with eukaryotic lipases (Falk et al. 1999). In addition, Arabidopsis PAD4, a gene required for expression of multiple defense responses after pathogen infection, encodes a lipase containing a lipase motif and the putative lipase catalytic triad (Jirage et al. 1999). However, the physiological roles of these enzymes in plant defense mechanisms remain unclear.
Colletotrichum gloeosporioides (Penz.) Penz. and Sacc. in Penz. is the causal agent of anthracnose diseases affecting fruit crops (Daykin 1984; Oh et al. 1998). The initial infection sequence of C. gloeosporioides consists of conidium germination and appressorium formation (Bailey et al. 1992). After that, the infection hypha is produced from the appressorium and penetrate into the host tissues to colonize the host. In the pepper-C. gloeosporioides pathosystem, only mature-unripe fruits showed susceptible interaction, while ripe fruits were resistant for interaction (Kim et al. 1999; Oh et al. 1998). A lower level of appressorium formation was observed in the resistant interaction in comparison to the susceptible interaction. This suggests that appressorium formation is positively linked to the development of anthracnose disease in this pathosystem.
The inventors previously cloned a pepper esterase gene (PepEST) that is highly expressed during the resistant interaction between the ripe fruit and C. gloeosporioides (U.S. Pat. No. 6,018,038; EP 1 018 554 A1). Here we have reported that recombinant PepEST protein expressed in Escherichia coli exhibited substrate specificity in hydrolyzing p-nitrophenyl esters. The recombinant PepEST inhibited appressorium formation of the anthracnose fungus in a dose-dependent manner, and protected pepper fruits against C. gloeosporioides infection. Finally, we propose that the recombinant PepEST affects a signal transduction pathway(s) involved in appressorium formation based on experimental results obtained with the rice blast fungus Magnaporthe grisea. 
This invention provides for a use of enzymes showing esterase activity for protection of plants against phytopathogenic fungi. Preferentially, the enzymes are esterase that can protect plants against phytopathogenic fungi by inhibition of fungal appressorium formation. The use of the esterases that can be from animals and plants can be provided for the purpose of plant protection. More preferably, the esterase is from the genus Solanum of the family Solanaceae. The esterase can be, for example, from a pepper plant. The fungi can be, for instance, pepper anthracnose fungus Colletotrichum gloeosporioides and rice blast fungus Magnaporthe grisea. 
A pepper esterase gene (PepEST) that is highly expressed during a resistant interaction between pepper (Capsicum annuum) and the anthracnose fungus Colletotrichum gloeosporioides has been previously cloned [The sequence of pepper esterase gene (PepEST) was disclosed in U.S. Pat. No. 6,018,038]. Glutathione-S-transferase tagged recombinant PepEST protein expressed in Escherichia coli showed substrate specificity for p-nitrophenyl esters [The sequence of pepper esterase protein (PepEST protein) was disclosed in U.S. Pat. No. 6,018,038].
Inoculation of susceptible-unripe pepper fruits with C. gloeosporioides spores amended with the recombinant PepEST protein did not cause anthracnose symptoms on the fruit. The recombinant protein has no fungicidal activity, but it significantly inhibits appressorium formation of the anthracnose fungus in a dose-dependent manner. An esterase from porcine liver also inhibited appressorium formation.
In addition, the recombinant PepEST protein inhibited appressorium formation in the rice blast fungus Magnaporthe grisea. Inhibition of appressorium formation in M. grisea by the recombinant protein was reversible by treatment with cAMP or 1,16-hexadecanediol. The results suggest that the recombinant protein regulates appressorium formation by modulating the cAMP-dependent signaling pathway in this fungus. Taken together, the PepEST esterase activity can inhibit appressorium formation of C. gloeosporioides, which may result in protection of the unripe fruit against the fungus